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Abstract:

Disclosed are a DNA aptamer specifically binding to human cardiac
troponin I, and a composition and a diagnostic kit for the diagnosis of
acute cardiovascular diseases, comprising the same. Being superior in
specificity and stability to antibodies which are conventionally used to
diagnose acute cardiovascular diseases, the DNA aptamers specifically
binding to human cardiac troponin I can be developed into biosensors
which determine human cardiac troponin I levels with high sensitivity and
accuracy, greatly contributing to the diagnosis in an early stage of
acute cardiovascular diseases. It is expected to lots of help for
increase of diagnostic accuracy.

Claims:

1. A DNA aptamer specifically binding to human cardiac troponin I,
wherein the DNA aptamer has a base sequence selected from the group
consisting of SEQ ID NOS: 1 to 6.

2. A composition for diagnosis of an acute cardiovascular disease,
comprising the DNA aptamer of claim 1.

3. A diagnostic kit for an acute cardiovascular disease, using the DNA
aptamer of claim 1.

Description:

CROSS-REFERENCE(S) TO RELATED APPLICATIONS

[0001] The present application claims priority of Korean Patent
Application Nos. 10-2011-0054795 and 10-2011-0145434, filed on Jun. 7,
2011 and Dec. 29, 2011, respectively, which is incorporated herein by
reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to a DNA aptamer specifically binding
to human cardiac troponin I, and a composition and a diagnostic kit for
the diagnosis of acute cardiovascular diseases, comprising the same.

[0004] 2. Description of the Related Art

[0005] Troponin is a complex of three regulatory proteins (troponin C,
troponin I and troponin T) that is attached to the protein tropomyosin
and lies within the groove between actin filaments in muscle tissue to
regulate the contraction and relaxation of muscle cells. When the
intracellular levels thereof rise, Ca2+ is bound to specific sites
on Troponin C to produce a conformational change in Troponin I so that
myosin can attach to the actin filament active sites, giving rise to the
contraction of the muscle. This action is observed in both skeletal and
cardiac muscles. The length of the amino acid sequence of Troponin I and
Troponin T that act on cardiac muscle are different from the
corresponding ones expressed in skeletal muscle.

[0006] The expression level of cardiac troponin I is known to rapidly
increase upon the outbreak of acute cardiovascular diseases. Thus, the
detection of this protein is very important for the initial diagnosis of
acute cardiovascular diseases.

[0007] An aptamer is a single strand DNA (ssDNA) or RNA (ssRNA) that binds
to a specific target. Thanks to their high affinity and stability to a
specific target, they have recently been extensively developed and
actively applied to the therapy and sensors for diagnosis of diseases.
The synthesis of aptamers can be relatively simple, and cells, proteins
and even small organic substance can be utilized as their targets, which
allows for the development of new detection methods. In addition,
aptamers find a wide range of applications in various fields, including
the development of therapeutics, drug delivery systems, biosensors for
diagnosis, etc. because their specificity and stability are superior to
those of the antibodies that were developed previously.

[0008] Antibodies developed for diagnostic use are prepared using the
immune system and thus suffer from the disadvantage of their preparation
consuming a lot of time and expense, comparatively. Further, they are
proteins that have poor stability, compared to aptamers, DNA or RNA,
which may act as an obstruction to the development of highly sensible
sensors.

[0009] Although many detection systems for troponin I have been developed
on the basis of antibodies to troponin I, as mentioned above, they are
subject to a lot of limitations. There is therefore a need for a
detection system that is more stable and which can be operated at low
cost and effectively diagnoses acute cardiovascular diseases in an early
stage.

SUMMARY OF THE INVENTION

[0010] The present invention is to provide a DNA aptamer specifically
binding to human cardiac troponin I, and a composition and a kit for the
diagnosis of an acute cardiovascular disease, comprising the same.

[0011] However, the technical objects to be achieved by the present
invention are not limited to those mentioned above and other objects may
be clearly understood by those skilled in the art from the description
given below.

[0012] In accordance with an aspect thereof, the present invention
provides an aptamer that specifically binds to human cardiac troponin I
which has a base sequence selected from the group consisting of SEQ ID
NOS: 1 to 6.

[0013] In accordance with another aspect thereof, the present invention
provides a composition for the diagnosis of an acute cardiovascular
disease, comprising a DNA aptamer specifically binding to human cardiac
troponin I.

[0014] In accordance with a further aspect thereof, the present invention
provides a diagnostic kit for an acute cardiovascular disease that uses a
DNA aptamer that specifically binds to human cardiac troponin I.

[0015] Superior in specificity and stability to antibodies which are
conventionally used to diagnose acute cardiovascular diseases, the DNA
aptamers specifically binding to human cardiac troponin I in accordance
with the present invention can be developed into biosensors which
determine human cardiac troponin I levels with sensitivity and accuracy,
greatly contributing to the diagnosis in an early stage of acute
cardiovascular diseases. It is expected to lots of help for increase of
diagnostic accuracy.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The above and other objects, features and other advantages of the
present invention will be more clearly understood from the following
detailed description taken in conjunction with the accompanying drawings,
in which:

[0017] FIG. 1 shows the expression of Troponin I protein as measured by
SDS PAGE.

[0038] To develop an aptamer as a substitute for an antibody to cardiac
troponin I, human cardiac troponin I was expressed in a bacterial
expression system, purified, and utilized to select DNA aptamers with the
SELEX (Systematic Evolution of Ligands by EXponential enrichment)
technique, which were then analyzed for sequence and structure,
culminating in the present invention.

[0039] In greater detail, the present invention provides a DNA aptamer
having the base sequence of one of SEQ ID NOS: 1 to 6 that specifically
binds to human cardiac troponin I.

[0040] Also, the present invention provides a composition and a kit for
the diagnosis of an acute cardiovascular disease, comprising a DNA
aptamer specifically binding to human cardiac troponin I.

[0041] In addition to the DNA aptamer, the composition of the present
invention may comprise pharmaceutically or physiologically acceptable
vehicles, excipients or diluents.

[0043] A better understanding of the present invention may be obtained
through the following examples which are set forth to illustrate, but are
not to be construed as limiting the present invention.

Example 1

Troponin I Gene Cloning

[0044] For use in the amplification of a human cardiac troponin I gene, a
5' primer with a BamH1 restriction site (GGATCC ATG GCGGAT GGG AGC AG;
SEQ ID NO: 7) and a 3' primer with a Hind3 restriction site (AAGCTT
TCAAAA CTT TTT CTT GCG G; SEQ ID NO: 8) were synthesized. For use in the
amplification of a human cardiac troponin T gene, a 5' primer with a
EcoRI restriction site (GAATTC ATG TCT GAC ATA GAA GAG GTG GTG; SEQ ID
NO: 9) and a 3' primer with a XhoI restriction site (CTCGAG CTA TTT CCA
GCG CCC GGT; SEQ ID NO: 10) were synthesized. And for use in the
amplification of a human cardiac troponin C gene, a 5' primer with a
EcoRI restriction site (GAATTC ATG GAT GAC ATC TAC AAG GCT GC; SEQ ID NO:
11) and a 3' primer with a XhoI restriction site (CTCGAG CTA CTC CAC ACC
CTT CAT GAA CTC; SEQ ID NO: 12) were synthesized. Using these primers,
amplification was conducted on the cDNA obtained from HEK293 cells in the
presence of i-pfu polymerase. In this regard, PCR was performed with 30
cycles of 1) denaturing the double strand of the template at 95°
C. for 1 min, 2) annealing the template with the primers at 58° C.
for 30 sec, and 3) extending new strands at 72° C. for 1 min.

[0045] The amplified human cardiac troponin I gene and troponin C gene
were digested with the restriction enzymes, ligated to a pET28a vector
containing (His)6-tag. The amplified human cardiac troponin T gene
was digested with the restriction enzymes, ligated to a pET21a vector.
Troponin I gene and Troponin C gene were transformed into BL21(DE3) E.
coli. For co-expression with troponin I, troponin T gene was transformed
into BL21-CodonPlus (DE3)-RIG E. coli.

Example 2

Expression of Troponin I Protein

[0046] The BL21(DE3) cells transformed with the human cardiac troponin I
gene were grown at 37° C. in an LB (Luria Bertani) medium to an
OD600 (optical density) of 0.6. Subsequently, the expression of the
protein was induced by incubating the cells at 18° C. for 16 hours
in the presence of 0.2 mM IPTG
(isopropyl-thio-β-D-galactopyranoside). The expression of the
protein was confirmed by SDS-PAGE (sodium dodecyl sulfate polyacrylamide
gel electrophoresis). After being harvested by centrifugation, the cells
were washed once with PBS (10 mM sodium phosphate, 150 mM NaCl, pH 7.4).
The SDS-PAGE results are shown in FIG. 1.

[0047] In FIG. 1, a marker (M) for indicating protein sizes was run on
lane 1, the protein obtained before IPTG induction (bf) on lane 2, and
proteins obtained after IPTG (af) on lanes 3 and 4. As can be seen in the
SDS PAGE of FIG. 1, Troponin I, was detected at 28 KD after the IPTG
induction.

Example 3

Expression of Troponin Complex

[0048] As expression of troponin I, T, BL21-CodonPlus (DE3)-RIG E. coli,
transformed with human cardiac troponin I gene and troponion T gene, and
BL21(DE3) cell transformed with troponin C gene were grown at 37°
C. in an LB (Luria Bertani) medium to an OD600 (optical density) of
0.6. Subsequently, the expression of the protein was induced by
incubating the cells at 18° C. for 16 hours in the presence of 0.2
mM IPTG (isopropyl-thio-β-D-galactopyranoside). The expression of
the protein was confirmed by SDS-PAGE (sodium dodecyl sulfate
polyacrylamide gel electrophoresis). After being harvested by
centrifugation, the cells were washed once with PBS (10 mM sodium
phosphate, 150 mM NaCl, pH 7.4). The SDS-PAGE results are shown in FIG.
2.

[0049] In FIG. 2, a marker (M) for indicating protein sizes was run on
lane 1, the protein obtained before IPTG induction (bf) on lane 2. As can
be seen in the SDS PAGE of FIG. 2, Troponin I, Troponin T and Troponin C
were detected after the IPTG induction.

Example 4

Purification of Troponin I Protein

[0050] To isolate the human cardiac troponin I protein expressed in the
bacterial cell BL21(DE3) to a high purity, the cells were lysed in a
lysis buffer (20 mM Tris, 500 mM NaCl, 0.5 mM mercaptoethanol, 3%
glycerol, 0.01% Tween 20, pH 8.0) and ruptured by sonication for 10 min.
Centrifugation at 15,000 rpm for 30 min separated proteins in a
supernatant from the cell debris.

[0051] The affinity of Ni-NTA (Ni-Nitrilo-triacetic acid) for the
(His)6-tag amino acid residues was used to isolate the protein to a
high purity. In this regard, FPLC (Fast protein liquid chromatography)
was coupled with an Ni-NTA column to which the supernatant containing
cardiac troponin I was then loaded. The target protein bound to the
column was eluted with elution buffer (20 mM Tris, 500 mM NaCl, 0.5 mM
β-mercaptoethanol, 3% glycerol, 0.01% tween 20,300 mM imidazole, pH
8.0) because the (His)6-tag of the protein competes with imidazole.

[0052] For additional purification, the fraction containing cardiac
troponin was subjected to size exclusion chromatography by gel filtration
using a Superdex75 column to obtain more pure protein. The imidazole used
was removed with the final buffer (20 mM Tris, 300 mM NaCl, 0.5 mM
β-mercaptoethanol, 3% glycerol, 0.01% tween 20, pH 8.0). The results
are shown in FIG. 3.

[0053] In FIG. 3, a marker for indicating protein sizes was run on lane 1
and the fraction obtained from the above-mentioned purification processes
was on lane 2. As can be seen in the SDS-PAGE of FIG. 3, Troponin of high
purity I was detected at 25 kD.

Example 5

Purification of Troponin Complex (Troponin T, C, I)

[0054] To isolate the troponin complex protein to a high purity, the
cells, were expressed troponin I and troponin T, were lysed in a lysis
buffer (20 mM Tris, 500 mM NaCl, 0.5 mM mercaptoethanol, 5% glycerol, pH
8.0) and ruptured by sonication. And soluble proteins were isolated by
Centrifugation.

[0055] As purification of troponin T and I proteins, the affinity of
Ni-NTA (Ni-Nitrilo-triacetic acid) for the (His)6-tag amino acid
residues was used to isolate the protein to a high purity. In this
regard, FPLC (Fast protein liquid chromatography) was coupled with an
Ni-NTA column to which the supernatant containing troponin T and troponin
I was then loaded. The target protein bound to the column was eluted with
elution buffer (20 mM Tris, 500 mM NaCl, 0.5 mM β-mercaptoethanol,
5% glycerol, 300 mM imidazole, pH 8.0).

[0056] For confirmation of troponin complex and measurement of SPR,
troponin C with a (His)6-tag was made. (His)6-tag of troponin I
and troponin T was eliminated by TEV (Tobacco Etch Virus) protease.
Specifically, for recognition of TEV protease behind (His)6-tag and
elimination, TEV protease and protein were performed at 20° C. for
6 hr.

[0057] Troponin I and troponin T were eluted by removing imidazole in
elution buffer with G-25 column. And then loaded Ni-NTA column, Purified
troponin I and troponin T were gotten by separating from (His)6-tag

[0058] Troponin C was purified by Ni-NTA column with containing
(His)6-tag and eluted from imidazole by G-25 column.

[0059] For getting of troponin complex, troponin C which contains purified
(His)6-tag was coupled with Ni-NTA, loaded supernatant containing
troponin T and troponin I, and then induced to formation of troponin
complex.

[0060] Troponin I and troponin T which were not coupled with Troponin C
were passed, troponin complex was gotten by elution buffer.

[0061] For additional purification, troponin complex was subjected to size
exclusion chromatography by gel filtration using a Superdex 200 column to
obtain more pure protein. The imidazole used was removed with the final
buffer (20 mM Tris, 300 mM NaCl, 0.5 mM β-mercaptoethanol, 5%
glycerol, pH 8.0). The results are shown in FIG. 4.

[0062] In FIG. 4, a marker for indicating protein sizes was run on lane 1
and the fraction obtained from the above-mentioned purification processes
was on lane 2.

Example 6

Search for Aptamers of Troponin I by SELEX

[0063] <6-1> Construction of ssDNA (Single-Stranded DNA) Library

[0064] A library of 90 sequences, each having primer sequences for PCR
amplification and cloning at opposite ends with a random DNA sequence of
40 bases between the primer sequences, was constructed
(5'-CACCTAATACGACTCACTATAGCGGATCCGA-N40-CTGGCTCGAACAAGCTTGC-3'; SEQ ID
NO: 13).

[0065] In addition, a 5' primer (5'-CACCTAATACGACTCACTATAGCGGA-3'; SEQ ID
NO: 14), a 3' primer (5'-GCAAGCTTGTTCGAGCCAG-3'; SEQ ID NO: 15) and a
biotin-conjugated 3' primer (5'-Biotin-GCAAGCTTGTTCGAGCCAG-3'; SEQ ID NO:
16) were used for PCR amplification and ssDNA production. All the
oligonucleotides used in the present invention were synthesized and
subjected to PAGE purification by Bionics (Korea).

[0067] The purified human cardiac Troponin I was immobilized to the
magnetic bead Dynabead (Invitrogen, Norway), which allows the His-tag to
bind to its cobalt-coated surface.

[0068] In this regard, the protein was fixed to the beads by washing 20
μL of the beads with a binding buffer (20 mM Tris, 300 mM NaCl, 3%
glycerol, 0.01% Tween 20, 5 mM MgCl, pH 8.0) by means of an external
magnet and incubating the beads with 150 μL of a binding buffer
containing 150 pmol of the protein.

[0070] To select aptamers specific for human cardiac troponin I, a
specific separation method using a magnet was conducted.

[0071] First, a library of the ssDNAs (1 nmol) was dissolved in 100 μL
of a binding buffer and was incubated at 90° C. for 3 min and then
at 4° C. for one hour to allow the ssDNA to form the most stable
conformation. Subsequently, this library was reacted for one hour with
the Troponin I protein immobilized to the magnetic bead, with gentle
agitation. Then, the beads were washed twice with the binding buffer to
remove the ssDNA which remained unbound to the Troponin I immobilized to
the beads.

[0072] Afterwards, the ssDNA was separated from the proteins bound
thereto. In this context, the ssDNA and the proteins bound thereto were
eluted with elution buffer (20 mM Tris, 300 mM NaCl, 3% glycerol, 0.01%
Tween 20, 5 mM MgCl2, 300 mM imidazole, pH8.0). The ssDNA eluate was
precipitated in ethanol, dissolved in 100 μL of distilled water, and
used as a template for PCR amplification using the 5' primer and the
biotin-conjugated 3' primer in the presence of i-pfu polymerase (Intron
Biotechnology, Korea). To isolate ssDNA for selection, the
biotin-conjugated PCR product was incubated for one hour with
streptavidin-coated magnetic beads in a coupling buffer (5 mM Tris-HCl, 5
mM EDTA, 1 M NaCl, 0.01%), followed by incubation with 100 μL of 100
mM NaOH for 5 min to separate only ssDNA. The ssDNA was obtained using an
external magnet.

[0073] The first selected ssDNA was used in subsequent repetitive
selection. For stringent selection, the amount of ssDNA and the
concentration of Troponin I were gradually decreased in subsequent
repetitions. During the selection process, the binding of ssDNA to
Troponin I was monitored by measuring the concentration of the ssDNA
eluted by the repeated selections with a UV spectrometer (Biochrom Libra
S22 spectrometer). The results are shown in FIG. 5.

[0074] FIG. 5 shows the extent of binding of the aptamers with the protein
that was immobilized to magnetic beads during the selection of the
aptamers. The extent of binding is expressed as a percentage (%) of the
concentration of the bound aptamer over the concentration of the aptamer
used. As can be shown in FIG. 5, the numbers of the aptamer DNA binding
to the protein increased with an increase in the selection round.

[0075] <6-4> Analysis of Sequence and Structure of Aptamers

[0076] The ssDNA selected in the 12th round was amplified by PCR
using the unmodified 5' and 3' primers and cloned to pENTR/TOPO (TOPO TA
Cloning kit, Invitrogen, USA) which was then transformed into E. coli
TOP10 (Invitrogen, USA). The clones harboring the ssDNA were purified
using a miniprep kit (GeneAll, Korea) and subjected to base sequencing
(COSMO Genetech, Korea). As a result, the ssDNA sequences were identified
as SEQ ID NOS: 1 to 6 and are listed in Table 1, below.

[0077] To analyze the structural similarity of the selected ssDNA, the
secondary structures of the selected ssDNA sequences were analyzed using
the Mfold program (http://mfold.bioinfo.rpi.edu/cgi-bin/dna-form1.cgi).
As shown in FIGS. 6 to 8, they were found to have a common stem-loop
structure attributable to the consecutive T-C sequence.

SPR (Surface Plasmon Resonance) Assay for Binding Strength between
Troponin I and Aptamer

[0078] SPR (surface Plasmon resonance) is a phenomenon occurring between
light and electrons on metal such as gold in which when a light with a
specific wavelength is incident on a metallic surface, most of the light
energy is transferred to free electrons of the metal, resulting in
resonance with the creation of evanescent wave. The binding strength can
be determined by measuring the resonance wavelength shift dependent on a
change in composition on the surface of the sample conjugated with the
metal. To determine binding strength between Troponin I and the aptamers,
SPR was measured using an Ni-NTA (Ni-Nitrilo-triacetic acid)-coated
surface chip (Biacore AB, Sweden).

[0079] The Troponin I protein (200 nM) was immobilized to the Ni-NTA chip
and then coupled with the aptamer DNAs. The aptamer DNAs were used at
concentrations of 25 nM, 50 nM, 100 nM, and 200 nM. For comparison, the
binding strength with cardiac Troponin I of a commercially available
antibody (Abcam, U.K.) was also measured. Its Kd was observed to be 20.1
nM which is higher than that of the aptamers of the present invention.

[0081] The binding strength with troponin complex of aptamers was measured
in the same way as above. The aptamer DNAs were used at concentrations of
50 nM, 100 nM, 200 nM and 400 nM. As a result, it was observed that
troponin complex combined to troponin I effectively. Its Kd was observed
which is higher than that of the binding troponin I or similar.

[0083] It is understood to a person skilled in the art that the above
description of the present invention is susceptible to various
modifications, changes and adaptations, and the same are intended to be
comprehended within the meaning and range of equivalents of the appended
claims. Therefore, the embodiments and attached drawings disclosed in the
present invention are not intended to limit the technical spirit of the
present invention, but are intended to describe the invention. The
technical spirit of the present invention is not limited to such
embodiments and drawings.